Human antibodies specifically binding to the hepatitis B virus surface antigen

ABSTRACT

Provided are antibodies specifically binding to the HBV surface antigen (HBsAg) which are effective for the prevention or treatment of hepatitis B.

CROSS REFERENCE TO RELATED APPLICATION

This application is a National Stage of International Application No.PCT/KR2010/004445, filed Jul. 8, 2010, claiming priority based on KoreanPatent Application No. 10-2009-0130900 filed Dec. 24, 2009, the contentsof all of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies specifically binding to thehepatitis B virus surface antigen (HBsAg).

BACKGROUND OF THE INVENTION

Hepatitis B virus (HBV) is a member of the Hepadnavirus family, andcauses acute and chronic hepatitis. About 350 million people of theworld's population, in particular, 5˜8% of the people in Korea andChina, are chronic HBV patients, and HBV is a main cause of liverdiseases and liver cancer. Although the development of a vaccine againstHBV has made it possible to prevent hepatitis B, but still many peopleare suffering from chronic hepatitis by HBV infection. The HBV infectioninduces hepatitis and liver cirrhosis as well as liver cancer, and theincidence of liver cancer in chronic hepatitis patients is 300 foldhigher than the normal. The WHO (World Health Organization) revealedthat about 80% of liver cancers result from chronic hepatitis B by HBVinfection.

Currently available therapeutic drugs for hepatitis B include nucleosideanalogues, such as lamivudine, adefovir dipivoxil, and others, and theyare known to inhibit HBV DNA polymerase. However, resistant virusesemerge in 75% of the patients after three-years of administrationtherewith, leading to decreased therapeutic efficacies, and thus, theyare used in combination with hepatitis B antibody agents so as toprevent vertical transmission or infection after liver transplantation.

The hepatitis B antibody agents currently used are prepared from humanblood sources having anti-HBV antibodies using highly technicalpurification and virus inactivation methods, but such methods cannotmeet the ever-rising demand due to the low availability of expensivehuman plasma with high anti-HBV antibody as well as the high cost ofinactivating the plausible human plasma-derived viruses.

Ever since a methodology of preparing a monoclonal antibody (mAb) wasestablished by Köhler and Milstein (1975), monoclonal antibodies derivedfrom mice have been mainly used for diagnosis or some treatment.However, the mouse antibody cannot be administered due to possiblegeneration of human anti-mouse antibody (HAMA) when applied to a humanbody for therapeutic purpose. In order to solve the problems of HAMA,there have been developed chimeric and humanized antibodies. Thechimeric antibody contains the variable regions (Fv fragment) of mousemAb which constitutes ˜30% of whole antibody molecule. In contrast, thehumanized antibody contains the CDRs (complementarity determiningregions) of mouse mAb which constitutes ˜10% of whole antibody molecule.Although such chimeric and humanized antibodies reduced the HAMAreaction significantly, it might be better to use human antibodies inthe treatment of chronic disease such as chronic hepatitis whichrequires a long-term and continuous administration.

The present invention has endeavored to develop novel, improvedantibodies and found that such antibodies can be used to inactivate HBVby binding to the HBV-surface antigen.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a novelantibody which specifically binds to hepatitis B virus surface antigen.

It is another object of the present invention to provide DNAs whichrespectively encode the heavy chain variable region and the light chainvariable region of said antibody, and an expression vector comprisingthe same.

It is still another object of the present invention to provide a cellline transformed with the expression vector.

It is a further object of the present invention to provide apharmaceutical composition for preventing or treating hepatitis B,comprising said antibody.

In accordance with one aspect of the present invention, there isprovided an antibody specifically binding to hepatitis B virus surfaceantigen (HBsAg), comprising: a) a heavy chain variable region having theamino acid sequence of SEQ ID NO: 1; b) a light chain variable regionhaving any of the amino acid sequences of SEQ ID NOs: 2, 3, and 4; c) aheavy chain constant region; and d) a light chain constant region.

In accordance with another aspect of the present invention, there isprovided a DNA which encodes the heavy chain variable region having theamino acid sequence of SEQ ID NO: 1 or the light chain variable regionhaving any of the amino acid sequences of SEQ ID NOs: 2, 3, and 4, andan expression vector comprising the same.

In accordance with a still another aspect of the present invention,there is provided a cell line transformed with said expression vector.

In accordance with a further aspect of the present invention, there isprovided a composition for preventing or treating hepatitis B,comprising said antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings, which respectivelyshow:

FIG. 1: a photographic result of electrophoresis (1% agarose gel)analysis exhibiting DNAs which respectively encode the inventive heavychain variable (VH) and the light chain variable regions (VL)synthesized by PCR;

FIG. 2: a map of the phage-display vector pKS4H, comprising the heavychain variable region and the light chain variable region of theinventive antibody;

FIG. 3: a diagram showing the process of selecting an antibody from anantibody library using the panning technique;

FIG. 4: amino acid sequences of the single chain variable fragments(scFv) of the inventive antibodies selected from an antibody library;

FIG. 5: a map of the expression vector for expressing the heavy chain ofthe human antibody of the present invention, HB48-33-Heavy-pRC13,HB48-35-Heavy-pRC13, or HB48-59-Heavy-pRC13;

FIGS. 6, 7, and 8: maps of expression vectors for expressing the lightchains of the human antibodies of the present invention,HB48-33-Light-pKC12, HB48-35-Light-pKC12, and HB48-59-Light-pKC12,respectively;

FIG. 9: SDS-PAGE analysis of the heavy chain and light chain expressedfrom the transformant;

FIG. 10: relative affinities of the human antibodies (HB48-33, HB48-35,and HB48-59) to the HBV surface antigen;

FIG. 11: a map of vector pHBV1.3-MBRI prepared from pcDNA3.1 byinserting a HBV DNA sequence, the vector being used in the preparationof a mouse model of acute hepatitis B;

FIG. 12: a graph showing the neutralizing powers of the inventive humanantibodies, HB48-33, HB48-35, and HB48-59, which are selected by in vivoefficacy test using a mouse model of acute hepatitis B. It was confirmedthat the HBV surface antigen (HBsAg) was expressed 24 hours afterinjecting the plasmid of FIG. 11, and that the surface antigen wasreduced by administration of the inventive antibodies. The surfaceantigen was measured by Genedia HBsAg ELISA 3.0 (Green Cross MS, Korea).

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention is described in detail.

The present invention provides an antibody specifically binding tohepatitis B virus surface antigen (HBsAg), comprising: a) a heavy chainvariable region having the amino acid sequence of SEQ ID NO: 1; b) alight chain variable region having any of the amino acid sequences ofSEQ ID NOs: 2, 3, and 4; c) a heavy chain constant region; and d) alight chain constant region. The antibody of the present invention ischaracterized by the efficacy in preventing or treating hepatitis B byspecifically binding to hepatitis B virus surface antigen to inactivateHBV.

The antibodies specifically binding to the HBV surface antigen may bepreferably selected by modification of a phage display method (Smith,Science, 228, 1315-1317, 1985; and Hoogenboom & Chames, Immunol Today,21, 371-378, 2000). In the phage display method, a gene (gene III)encoding a surface protein of filamentous phage (e.g. M13, Fd or Fl) isfused with a gene encoding an antibody of interest, to produce virusparticles having a fused antibody exposed on the surface which is of anantibody-phage form. Subsequently, an antibody of interest can beselected from a phage library through the biopanning technique usinghigh specificity and affinity of the antibody and high infectiveproperty of the phage (Burton & Barbas, Adv. Immunol., 57, 191-280,1994; Winter et al., Annu. Rev. Immunol., 12, 433-455, 1994; andHoogenboom et al., Immunotechnology, 4, 1-20, 1998; Kim et al., HybridHybridomics, 21, 385-392, 2002). The phage display vector may be pKS4H(see Korean Patent No. 0635370) or pCANTAB5E, preferably, pKS4H.

The present inventors have selected three human antibodies (HB48-33,HB48-35, and HB48-59) from a phage library, and then examined theaffinity of antibodies and neutralizing ability against the HBV surfaceantigen (FIGS. 10 and 11). Additionally, amino acid sequences of thevariable regions of heavy and light chains were analyzed, and then itwas confirmed that all heavy chain variable regions have the amino acidsequence of SEQ ID NO: 1 and light chain variable regions have the aminoacid sequences of SEQ ID NO: 2, 3, and 4, respectively.

The constant regions of heavy chain and light chains of the inventiveantibodies may be identical to those of a human antibody.

The present invention provides a DNA encoding the antibody heavy chainvariable region having the amino acid sequence of SEQ ID NO: 1.Preferably, the DNA may comprise the polynucleotide having thenucleotide sequence of SEQ ID NO: 5 encoding the amino acid sequence ofSEQ ID NO: 1.

The present invention provides a DNA encoding the antibody light chainvariable region having any of the amino acid sequences of SEQ ID NOs: 2,3, and 4. Preferably, the DNA may comprise the polynucleotide having anyof the nucleotide sequences of SEQ ID NOs: 6, 7, and 8 encoding theamino acid sequences of SEQ ID NOs: 2, 3, and 4.

The present invention provides an expression vector for expressing theheavy chain variable region of the antibody specifically binding to theHBV surface antigen, comprising the DNA encoding the heavy chainvariable region of the antibody. Preferably, the expression vector maybe “HB48-33-Heavy-pRC13”, “HB48-35-Heavy-pRC13”, or“HB48-59-Heavy-pRC13” whose map is depicted in FIG. 5.

Specifically, the vector may be prepared by inserting the VH fragment(HB48VH) of the antibody selected by panning process into a suitablevector, e.g., pRC13 vector (deposit No. KCLRF-BP-00054; Korean PatentNo. 523732).

The present invention provides an expression vector for expressing thelight chain variable region of the antibody specifically binding to theHBV surface antigen, comprising the DNA encoding the light chainvariable region of the antibody. Preferably, the expression vector maybe “HB48-33-Light-pKC13” whose map is depicted in FIG. 6,“HB48-35-Light-pKC13” whose map is depicted in FIG. 7, or“HB48-59-Light-pKC13” whose map is depicted in FIG. 8.

Specifically, the vectors may be prepared by inserting each VL fragment(HB48-33VL, HB48-35VL, or HB48-59VL) of the antibodies selected bypanning processes into a suitable vector, e.g., pKC12 vector (depositNo. KCLRF-BP-00054; Korean Patent No. 523732).

The present invention provides an animal cell line transformed with theexpression vectors for expressing the variable regions of heavy chainand light chains of the inventive antibody. The animal cell line may beCHO (Chinese hamster ovary), HEK 293, or NSO cell line, preferably, CHO(Chinese hamster ovary) cell line.

The antibodies according to the present invention may be prepared bywhich the variable regions of heavy and light chains are linked to eachother.

The affinity of the inventive antibodies to the antigen may be measured,e.g., by the competitive ELISA (Kim et al., Hybridoma, 20, 265-272,2001). As shown in FIG. 10, the affinity of HB48-35 was highest amongthe human antibodies of the present invention, whereas those of HB48-59and HB48-33 were approximately 1.3 and 4.0 fold lower than HB48-35,respectively. Further, in an HBV mouse model, which is prepared byhydrodynamic injection of HBV DNA into C57BL6 mouse to induce hepatitisB-like symptoms (Hydrodynamic injection; Liu et al., Gene Therapy, 6,1258-1266, 1999), HB48-33, HB48-35, and HB48-59 antibodies showed theneutralizing ability of HBV in which surface antigen of HBV was reducedto a ground level by the antibody in a mouse blood (FIG. 12). Therefore,the antibodies of the present invention may be used for preventing ortreating hepatitis B by binding to HBV surface antigen to inactivateHBV.

In view of the result, the present invention provides a pharmaceuticalcomposition for preventing or treating hepatitis B, comprising theantibody. The composition of the present invention may be prepared intoa pharmaceutical formulation in accordance with conventional methods. Inpreparation of the formulation, the antibody is preferably mixed with acarrier or diluted with a carrier, or incorporated within a carrierwhich may be in the form of a container. When the carrier serves as adiluent, it may be a solid, semi-solid, or liquid which acts as avesicle, excipient, or medium for the antibody. Thus, the formulationmay be in the form of tablets, pills, powders, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft andhard gelatin capsules, sterile injectable solutions, sterile powers, andthe like. Examples of a suitable carrier, excipient, or diluents includelactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate,cellulose, methyl cellulose, microcrystalline cellulose,polyvinylpyrrolidone, water, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Theformulations can additionally include filling agents, anticoagulants,lubricating agents, wetting agents, flavoring agents, emulsifyingagents, preserving agents, and the like. The antibody composition of thepresent invention can further comprise interferons, anti-HBV monoclonalantibodies, anti-HBV polyclonal antibodies, nucleoside analogues, DNApolymerase inhibitors, siRNA agents, or vaccines for treatment, as ananti-viral agent, in conjunction with the antibody.

The HBV infection and HBV-associated diseases can be prevented ortreated by administering the inventive composition to a mammal includinga human. The dosage of the antibody within the composition will dependon the subject, the severity of the disease condition, administrationrate, and physician's determination. The antibody as an effectiveingredient can be administered to a mammal via a parenteral route in aneffective amount ranging from about 0.001 to 10 mg/kg body weight,preferably 0.005 to 1 mg/kg body weight per day, in a single dose or individed doses. In some cases, a small or large quantity of dose may besuitable, relative to the aforementioned dose, and the large quantity ofdose can be administered in divided small doses per day.

The following Examples are given for the purpose of illustration only,and are not intended to limit the scope of the invention.

EXAMPLE 1 Isolation of RNA

In order to select antibodies specifically binding to the HBV surfaceantigen, a human antibody library was constructed. A mixture of RNAsfrom human bone marrow, human thymus, human spleen and human B cell wasused. All RNAs except for human B cell RNA were purchased from Clontech(U.S.) and human B cell RNA was isolated as follows:

50 mL of blood taken from a healthy adult was diluted by mixing with 50mL of PBS. 3 mL of Ficoll-Paque PLUS (GE Healthcare, U.S.) was put in a15 mL tube and 4 mL of the diluted blood was added thereto. The mixturewas centrifuged at 3,000 rpm to isolate a white blood cell. 2 mL of theisolated white blood cell was mixed with 6 mL of PBS and centrifuged at100×g for 10 min. 100 μL of the white blood cell was mixed with 1 mL ofTrizole (Life Technology, U.S.) to isolate RNA.

Meanwhile, the isolated RNA was diluted with distilled water, and theabsorbance at 260 nm was measured to calculate its amount (1.8 μg/μL;Ultraspec 2000 UV-VIS spectrophotometer, GE, U.S.). Detailed procedureis as follows:

1 mL of trizole was added to 100 μL of white blood cell, shook well, andleft at room temperature (15° C. to 30° C.) for 5 min. Then, 200 μL ofchloroform was added, shook vigorously for 15 sec, and left at roomtemperature for 3 min. Subsequently, the mixture was centrifuged under acondition of 2-8° C., 15 min and 15,000 rpm, and the supernatant wastransferred into a new tube. 500 μL of isopropyl alcohol was added andmixed well, and left at room temperature for 10 min. Then, the mixturewas centrifuged at 2˜8° C. and 15,000 rpm for 5 min to remove thesupernatant. 1 mL of 75% ethanol was added thereto and the mixture wascentrifuged under a condition of 2˜8° C., 5 min and 15,000 rpm to removeethanol, and the RNA pellet was dried at room temperature for 5 min. 150μL of distilled water was added thereto to suspend the RNA pellet, andthe absorbance of the suspension was measured at 260 nm. The remnant wasstored at −20° C.

EXAMPLE 2 Amplification of Antibody Genes

1 μg of RNA isolated in Example 1 and 1 μL of pd(T)₁₂₋₁₈ (0.5 μg/μL)were mixed with distilled water to make final volume into 12.5 μL. Themixture was subjected to a reaction at 70° C. for 2 min and cooled inice. Then, 5× reaction buffer, 10 mM dNTP mix, recombinant RNaseinhibitor and MMLV reverse transcriptase (Clontech, U.S.) were addedthereto to make final volume into 20 μL, followed by the reaction at 42°C. for 1 hr and at 95° C. for 5 min to synthesize cDNA. PCR reaction wascarried out using LiquiMix QM Premix, Magenta (Neurotics Inc, Korea), 4μL of cDNA as a template, 19 μL of distilled water, and 1 μL (25μmol/μL) of primers designed for scFv region, heavy chain variableregion and light chain variable region (kappa and lambda), respectively[ScFv: SEQ ID NOs: 9 (Forward), 10 (Reverse-κ), and 11 (Reverse-λ);heavy chain variable region: SEQ ID NOs: 12 (Forward-VH1), 13(Forward-VH2), 14 (Forward-VH3), 15 (Forward-VH4), 16 (Forward-VH5), 17(Forward-VH6), 18 (Forward-VH7), 19 (Reverse); light chain variableregion κ chain: SEQ ID NOs: 20 (Forward-VK1/3A), 21 (Forward-VK1/3B), 22(Forward-VK2), 23 (Forward-VK4), 24 (Forward-VK5), 25 (Forward-VK6), 26(Reverse-JK-A), 27 (Reverse-JK-B), and 28 (Reverse-JK-C); light chainvariable region λ chain: SEQ ID NOs: 29 (Forward-VL1˜3-A), 30(Forward-VL1˜3-B), 31 (Forward-VL4), 32 (Forward-VL5), 33 (Forward-VL6),34 (Forward-VL7), 35 (Forward-VL8), 36 (Forward-VL9), 37 (Forward-VL10),38 (Reverse-JL-A), and 39 (Reverse-JL-B).

PCR reaction was carried out at 95° C. for 5 min, 95° C. for 1 min, 55°C. for 2 min, 72° C. for 2 min with 30 cycles, finally 72° C. for 15min.

The amplified antibody DNAs were identified by electrophoresis in 1.2%agarose gel (FIG. 1), and purified using Qiagen kit (Qiagen, Germany).As shown in FIG. 1, 350 bp of DNA bands corresponding to variableregions of the heavy and light chains (kappa and lambda) were obtained.In FIG. 1, M refers to a size marker, VH: heavy chain variable region(lane 1: heavy chain variable region type I; lane 2: heavy chainvariable region type II; lane 3: heavy chain variable region type III;lane 4: heavy chain variable region type VI; lane 5: heavy chainvariable region type V; lane 6: heavy chain variable region type VI; andlane 7: heavy chain variable region type VII), and VL to light chainvariable region (lane 9: light chain variable region 1/3κ; lane 10:light chain variable region 2κ; lane 11: light chain variable region 4κ;lane 12: light chain variable region 5κ; lane 13: light chain variableregion 6κ; lane 15: light chain variable region 1˜3λ; lane 16: lightchain variable region 4λ; lane 17: light chain variable region 5λ; lane18: light chain variable region 6λ; lane 19: light chain variable region7λ; lane 20: light chain variable region 8λ; lane 21: light chainvariable region 9λ; and lane 22: light chain variable region 10λ).

EXAMPLE 3 Restriction Enzyme Digestion of Antibody DNAs

VH and VL (kappa and lambda) prepared in Example 2 were digested withrestriction enzymes SfiI/BspEI and BspEI/NotI, respectively, and thedigested fragments were isolated from 1.2% agarose gel electrophoresisand purified using Qiagen kit.

EXAMPLE 4 Ligation of the Antibody DNAs and Preparation of Libraries

Phage-display vector pKS4H (Green cross Corp., Korea, see Korean PatentNo. 0635370) were digested using a restriction enzyme, SfiI/BspEI, andwas separated using 1.2% agarose gel electrophoresis, followed bypurification using Qiagen kit. 40 μg of the pKS4H was mixed with 10 μgof VH prepared in Example 3, and T4 DNA ligase (New England BioLabs,U.S.) was added thereto, followed by the reaction overnight at 25° C.The ligation mixture was purified using Qiagen kit, and was transformedinto E. coli XL1-blue (Stratagene, U.S.) by electroporation. Thetransformant was cultured in 100 mL of medium overnight, and the plasmidwas isolated. The plasmid was designated as “pKS4H-VH-ΔVL”.

The plasmid, pKS4H-VH-ΔVL, was digested with a restriction enzyme,BspEI/NotI, and purified as described above. Then, 40 μg of pKS4H-VH-ΔVLplasmid was mixed with 10 μg of VL PCR DNA prepared in Example 3 and T4DNA ligase (New England BioLabs, U.S.), and was subjected to a reactionovernight at 25° C. The ligation mixture was purified using Qiagen kit,and was transformed into E. coli XL1-blue by electroporation. Thetransformant was cultured in 100 mL of medium containing carbenicillinand tetracyclin at 37° C. for 2 hours. Then, M13 helper phage(Stratagene, U.S.) was inoculated to the medium and cultured for 16 hrto prepare a phage library as reported in Engberg et al (Mol.Biotechnol., 6, 287-310, 1995). Meanwhile, a plasmid was isolated fromthe E. coli, and designated as “pKS4H-VH-VL”. The map of the plasmid isdepicted in FIG. 2.

EXAMPLE 5 Selection of Antibodies Binding to the HBV Surface Antigen

Antibodies binding to the HBV surface antigen were selected by amodification of panning technique (Engberg et al., Mol. Biotechnol., 6,287-310, 1996; and Kim et al., Gene, 241, 19-25, 2000). Specifically,HBV surface antigen (Green cross Corp., Korea) was diluted with PBS andeach immunotube (NUNC, Denmark) was coated with the diluted antigen.Then, the phage library prepared in Example 4 was added to the coatedimmunotube and incubated for 2 hr at 37° C. Phages binding to HBV wereeluted using 0.1M of glycine buffer (pH 2.0). Subsequently, E. coliXL1-blue was infected with the phages and a helper phage was added. TheE. coli was incubated overnight and a PEG solution (20% PEG 8,000 and15% NaCl) was added thereto. Then, precipitated phages were collected(phage rescue) and the phages were again subjected to a reaction withthe immunotube coated with the HBV surface antigen, and the aboveprocedure was repeated 4 times. Three human antibodies binding to HBVsurface antigen were selected from the procedure, and designated asHB48-33, HB48-35, and HB48-59. The process of selecting human antibodiesusing phage-display libraries was illustrated in FIG. 3.

Each colony from 4^(th) panning was grown in 2 mL of medium, inaccordance with a known method (Kim et al., Gene, 241, 19-25, 2000), andthe expression of antibody was induced by treatment of IPTG (isopropylβ-D-1-thiogalactopyranoside). The expression of antibody was measured byELISA (Enzyme-Linked ImmunoSorbent Assay), using a 96-well plate coatedwith HBV surface antigen.

EXAMPLE 6 Sequence Analysis of Selected Antibodies

Colonies which secrete human antibodies HB48-33, HB48-35, and HB48-59selected in Example 5 were grown overnight in 10 mL of LB mediumcontaining 50 μg/mL of carbenicillin and plasmids were isolated usingQiagen plasmid mini kit (Qiagen, Valencia, Calif., U.S.) therefrom. Theplasmids were digested with SfiI/NotI, and electrophoresed in agarosegel so as to identify the insertion of fragments of antibodies. The DNAsequence of scFv inserted into the plasmid was analyzed. The nucleotidesequences of scFv were analyzed with a sequencing primer, p033 of SEQ IDNO: 40 in Genotech (Daejeon, Korea).

The DNA sequences of scFv of human antibodies HB48-33, HB48-35, andHB48-59 were translated into amino acids using a web-based program(www.expasy.org: DNA to Protein translate tool), and the translatedamino acid sequences were shown in FIG. 4. As shown in FIG. 4, humanantibodies HB48-33, HB48-35, and HB48-59 had heavy chain variableregions represented by same amino acid sequences, and light chainvariable regions represented by different amino acid sequences.

EXAMPLE 7 Construction of Expression Vectors

In order to convert the antibody fragments into intact immunoglobulins,antibody expression vectors, pRC13 and pKC12 were used (see KoreanPatent No. 523732; Deposit No. KCLRF-BP-00054).

The heavy chain expression vector pRC13 is a vector in which a VHfragment of an antibody can be easily inserted into HindIII and ApaIsites. As exemplified in FIG. 5, the DNAs encoding the heavy chainvariable regions of the human antibodies HB48-33, HB48-35, and HB48-59were amplified by PCR using primers of SEQ ID NOs: 41 (Forward) and 42(Reverse) under a condition of 95° C. for 1 min, 55° C. for 2 min, 72°C. for 2 min, digested with HindIII/ApaI, and inserted into pRC13 whichwas digested with same restriction enzymes. The recombinant vector wasdesignated “HB48-33-Heavy-pRC13”, “HB48-35-Heavy-pRC13”, or“HB48-59-Heavy-pRC13” (see FIG. 5).

Meanwhile, the light chain expression vector pKC12 is a vector in whicha VL fragment of an antibody can be easily inserted into NheI and ApaIsites. As exemplified in FIGS. 6, 7, and 8, DNAs encoding the K lightchain variable region of the human antibodies HB48-35 and HB48-59 wereamplified by PCR using primers of SEQ ID NOs: 43 (Forward) and 44(Reverse) under a condition of 95° C. for 1 min, 55° C. for 2 min, 72°C. for 2 min, and DNA encoding the λ light chain variable region of thehuman antibody HB48-33 was amplified by PCR using primers of SEQ ID NOs:43 (Forward) and 45 (Reverse) under a condition of 95° C. for 1 min, 55°C. for 2 min, 72° C. for 2 min. The amplified DNAs were digested withNheI/ApaI, and inserted into pKC12 which was digested with samerestriction enzymes. The recombinant vectors were designated“HB48-33-Light-pKC12”, “HB48-35-Light-pKC12”, and “HB48-33-Light-pKC12”,respectively (see FIGS. 6, 7, and 8).

EXAMPLE 8 Construction of Animal Cell Lines Secreting Antibodies

2×10⁵ CHO (Chinese hamster ovary) cells were grown in T-25 flask (NUNC,Denmark) filled with α-MEM medium (Life Technologies, U.S.) containing10% FBS (Life Technologies, U.S.), in 37° C. incubator in the presenceof 5% CO₂, 24 hours prior to transfection, until confluency reaches 50%.Then, 30 μg of lipofectin (Life Technologies, U.S.) was added to 1.5 mLof opti-MEM (Life Technologies, U.S.) and left undisturbed for 90 min atroom temperature. At the same time, 8 μg of HB48-33-Heavy-pRC13 & 7 μgof HB48-33-Light-pKC12, 8 μg of HB48-35-Heavy-pRC13 & 7 μg ofHB48-35-Light-pKC12, and 8 μg of HB48-59-Heavy-pRC13 & 7 μg ofHB48-59-Light-pKC12 were mixed respectively and added to 15 mL ofopti-MEM, then left undisturbed for 90 min at room temperature. After 90min, the medium containing lipofectin was mixed with the mediumcontaining HB48-33-Heavy-pRC13 & HB48-33-Light-pKC12,HB48-35-Heavy-pRC13 & HB48-35-Light-pKC12, and HB48-59-Heavy-pRC13 &HB48-59-Light-pKC12, respectively, and incubated for 15 min at roomtemperature. During the reaction, the medium was removed from the cells,and the cells were washed three times with PBS for transfection. Thereaction mixture was added to the washed cells and incubated for 6hours. After 6 hours, the reaction mixture was removed, and α-MEM mediumwas added and incubated for 48 hours. Then the cells were treated withtrypsin (Life Technologies, U.S.) to detach from the flask, diluted withα-MEM medium, and subcultured at 96-well plate (NUNC, Denmark). At thetime, the α-MEM medium contains no ribonucleoside anddeoxyribonucleoside, whereas contains 10% of dialyzed FBS (LifeTechnologies, U.S.) and 550 μg/mL of G418 (Sigma, U.S.). The medium wasreplaced with a fresh medium every two days. The culture supernatantforming colonies was collected for ELISA analysis, and selected cellswere transferred into 12-well plate. When the cells grew well in 12-wellplate, the cells were transferred into 6-well plate, and when the cellsalso grew well in E-well plate, methotrexate (MTX, Choongwae PharmaCorporation, Korea) was treated thereto. The initial concentration ofMTX was 20 nM, and increased to 80 nM, 320 nM and 1 μM according to theadaptability of cells. Cell lines which survived at a concentration of 1μM and had a high amount of antibody secretion were selected. Theselected cell lines were mass-cultured in an incubator with 65 rpm, 5%CO₂ and 37° C., using spinner flask and serum-free medium. 10⁸ cellswere cultured in 250 mL flask filled with 100 mL of serum-free medium.When the number of the cells became 2 times of the initial inoculation,cells were collected by centrifugation at 1,000 rpm for 5 min. Thecollected cells were cultured again in 500 mL flask filled with 200 mLof medium. When the number of the cells became 2 times the initialinoculation, cells were collected by centrifugation at 1,000 rpm for 5min, and transferred into 3 L spinner flask filled with 1 L of medium.Sodium butyrate (Aldrich, U.S.) was added thereto to a finalconcentration of 2 mM, the cells were cultured for 5 days, and thesupernatant was collected from the medium. From supernatant collectedfrom spinner flasks, antibodies were purified using a protein A-agarosecolumn (Amersham Pharmacia Biotech, U.S.) and were analyzed by SDS-PAGE.

As shown in FIG. 9, about 50 kDa of heavy chain bands and 25 kDa oflight chain bands were observed, indicating that antibodies weresynthesized correctly.

EXAMPLE 9 Measurement of Antibody Affinity

The affinities of the antibodies obtained in Example 8 to the HBVsurface antigen were determined by a competitive ELISA method (Kim etal., Hybridoma, 20, 265-272, 2001), and the results were shown in FIG.10. Brief procedure is as follows:

(1) Determination of Optimum Concentration of Antibodies

A. Preparation of a Plate

100 μL of HBV surface antigen (Green cross Corp., Korea) at a 2 μg/mLdilution in PBS was added to each well of an ELISA plate and incubatedovernight at 4° C. Each well of the plate was washed once with PBST, 300μL of 1% BSA-PBS solution was added to each well, and incubated for 1hour at room temperature.

B. 1^(st) Reaction

100 μL of each purified antibody (0.5 μg/mL) was added to each well ofplate, and incubated for 2 hours at room temperature, and washed fourtimes with PBST.

C. 2^(nd) Reaction

100 μL of goat anti-human IgG (Fab specific)-perxoidase conjugate(Sigma) at a 1:5000 dilution in 1% BSA-PBS was added to each well,incubated for 1 hour at room temperature, and washed four times withPBST.

D. Substrate Reaction

100 μL of TMB (3,3′,5,5′-tetramethylbenzidine, Microwell peroxidasesubstrate system (KPL, MD, U.S.) was added to each well and O.D valuewas measured at 405 nm. Optimum concentration of antibody was determinedas the antibody concentration that gives half-maximum binding.

(2) Competitive ELISA

A. Preparation of a Plate

100 μL of HBV surface antigen (Green cross Corp., Korea) at a 2 μg/mLdilution in PBS was added to each well of an ELISA plate and incubatedovernight at 4° C. Each well was washed once with PBST, 300 μL of 1%BSA-PBS solution was added to each well, and incubated for 1 hour atroom temperature.

B. 1^(st) Reaction

2 μg of HBV surface antigen was diluted by a two-fold serially and 10 μLof the diluted HBsAg was added to each well of the plate. Then, 90 μL ofthe antibody diluted to the optimum concentration determined in (1) wasadded to each well, incubated for 2 hours at room temperature, andwashed 4 times with PBST.

C. 2^(nd) Reaction

100 μL of goat anti-human IgG (Fab specific)-perxoidase conjugate(Sigma) at a 1:5,000 dilution in 1% BSA-PBS was added to each well,incubated for 1 hour at room temperature, and washed four times withPBST.

D. Substrate Reaction

100 μL of TMB (3,3′,5,5′-tetramethylbenzidine, Microwell peroxidasesubstrate system (KPL, MD, U.S.) was added to each well and O.D valuewas measured at 405 nm. The concentration of HBsAg which inhibits 50% ofmaximum binding (O.D value in which no competing EGFR exists) wasdetermined as Kd.

As shown in FIG. 10, the affinity of HB48-35 was highest among the humanantibodies of the present invention, and in contrast those of HB48-59and HB48-33 were approximately 1.3 and 4.0 fold lower than HB48-35,respectively.

EXAMPLE 10 In Vivo Efficacy Test Using a Mouse Model of Acute HepatitisB

The neutralizing abilities against HBsAg of HB48-33, HB48-35, andHB48-59 were compared, in a C57BL6 mouse model which is prepared byhydrodynamic injection of HBV DNA to manifest hepatitis B-like symptoms.

Twenty female C57BL6 mice of 4 weeks old and weighing about 20 g, werepurchased from Charles Liver Laboratory (MA, U.S.), and were dividedinto 4 groups of 5 mice as shown in Table 1.

TABLE 1 Group Number of mice Test material and route Dosage Controlgroup 5 PBS, intravenous injection 0.2 mL Exp. group I 5 HB48-33 0.1 mg,0.2 mL intravenous injection Exp. group II 5 HB48-35 0.1 mg, 0.2 mLintravenous injection Exp. group III 5 HB48-59 0.1 mg, 0.2 mLintravenous injection

20 μg of vector pHBV-MBRI constructed by inserting HBV DNA into pcDNA3.1(Invitrogen, U.S.) (Shin et al., Virus Research 119, 146-153, 2006; SeeFIG. 11) were injected into all mice via mouse tail veins in 9.5% ofweight by a volume in a speed of 0.3 mL/min to induce an acute hepatitisB. After 24 hours, 0.2 mL of test materials listed in Table 1 wereinjected via mice tail veins. Serums were isolated at pre-injection (0hr), 24 hour post-injection, and 48 hour post-injection. The isolatedserums were diluted by 10 fold with goat serum, and then the bloodconcentrations of HBsAg were measured using Genedia HBsAg ELISA 3.0(Green Cross MS, Korea) and the results are shown in FIG. 12. As shownin FIG. 12, the blood concentration of HBsAg was maintained at maximumpoint for 48 hours, in the control group intravenously injected withPBS. In contrast, the blood concentration of HBsAg was not detected for24 to 48 hours, in groups treated with 0.1 mg HB48-33 (experimentalgroup I), 0.1 mg HB48-35 (experimental group II), and 0.1 mg HB48-59(experimental group III). Accordingly, it was confirmed that theantibodies of the present invention are very effective in neutralizingthe HBV surface antigen.

While the invention has been described with respect to the abovespecific embodiments, it should be recognized that various modificationsand changes may be made to the invention by those skilled in the artwhich also fall within the scope of the invention as defined by theappended claims.

What is claimed is:
 1. A human antibody specifically binding tohepatitis B virus surface antigen, comprising: a) a heavy chain variableregion having the amino acid sequence of SEQ ID NO: 1; b) a light chainvariable region having any of the amino acid sequences of SEQ ID NOs: 2,3, and 4; c) a heavy chain constant region; and d) a light chainconstant region.
 2. A pharmaceutical composition comprising the antibodyof claim 1 and a pharmaceutically acceptable carrier.